The capability for detecting microscopic particles has been proceeding toward smaller particles. For many applications it is essential that microscopic particles be detected in a liquid-phase environment. Existing techniques, usable in a liquid-phase environment, are based on optical trapping and on flow separation using hydrodynamically focused flows. Molecular identification by laser-induced fluorescence has been used with hydrodynamically focused flows to permit the detection of large and highly fluorescent molecules using conventional photomultiplier tubes to detect the molecule fluorescence.
Optical trapping and manipulation of viruses and bacteria are taught in A. Ashkin et al., "Optical Trapping and Manipulation of Viruses and Bacteria," Science 235, 1517 (1987). Rayleigh- and Mie-sized particles, i.e., a particle size range from about 10 .mu.m down to a few angstroms, have been trapped using optical forces to confine the particles. The only method of identification taught by Ashkin et al. appears to be a size determination from a scattering comparison with a sphere of known size. Further, a large number of particles are trapped.
A hydrodynamically focused flow system is taught by D. C. Nguyen et al., "Ultrasensitive Laser-Induced Fluorescence Detection in Hydrodynamically Focused Flows," J. Opt. Soc. Am. B4, 138 (1987), and D. C. Nguyen et al., "Detection of Single Molecules of Phycoerythrin in Hydrodynamically Focused Flows by Laser Induced Fluorescence," Anal. Chem. 59, 2158 (1987), incorporated herein by reference. As taught therein, improvements in the optics and reductions in the size of the probe volume provide a sensitivity effective to detect a single species containing the fluorescence equivalent of eight rhodamine-6G chromophores. The detection of single molecules of the highly fluorescent species phycoerythrin is reported.
A variety of modifications are reported to enhance the detection sensitivity of the device, with the improvements being related to conventional optics and flow dynamics, and with a sample volume reduction from 11 pL to 0.6 pL producing a concomitant reduction in detected background radiation. The reported sensitivities do not, however, enable the device to detect individual molecules that might typically be of interest, such as fluorophore-tagged versions of the base molecules that make up the DNA polymer.
Thus, available methods and apparatus for detecting particles in a flow stream do not provide the sensitivity for detecting individual molecules that might typically be encountered in immunofluorescence assay, flow cytometry, liquid chromatography, and similar applications. An agglomeration of molecules might be detected, but single molecules could not then be identified. This lack of capabilities in the art is overcome by the present invention and improved method and apparatus are provided for detecting a single modestly fluorescent molecule.
Accordingly, it is an object of the present invention to reliably detect a single fluorescent molecule.
Another object is to reliably detect single fluorescent molecules with a fluorescence equivalent to flouorescently-labeled versions of the bases forming the DNA polymer.
Yet another object is to provide an increased capability of rejecting background radiation.
One other object is to minimize the resolution limitations inherent in conventional optics while maintaining a large field of view.